Abstract
In response to alpha-melanocyte-stimulating hormone (alpha-MSH) or cAMP-elevating agents (forskolin and isobutylmethylxanthine), mouse B16 melanoma cells underwent differentiation characterized by increased melanin biosynthesis. However, the mechanism(s) underlying the regulation of melanogenesis during differentiation has not yet been clearly understood. Phospholipase D (PLD) has been reported to be involved in differentiation. This enzyme cleaves phosphatidylcholine upon stimulation with stimuli to generate phosphatidic acid. In the current study, the involvement of PLD in the regulation of melanogenesis characteristic of differentiation was examined using mouse B16 melanoma cells. Treatment of B16 cells with alpha-MSH was found to cause marked decreases in the PLD1 activity concurrent with its reduced protein level. Moreover, treatment of exogenous bacterial PLD also inhibited alpha-MSH-induced melanogenesis. To further investigate the role of PLD1 in the regulation of melanogenesis, we examined the effects of overexpression of PLD1 on melanogenesis in B16 melanoma cells. The B16 cells overexpressing PLD were prepared by transfection with the vector containing the cDNA encoding PLD1. The melanin contents in PLD1-overexpressing cells (B16/PLD1) were observed to be lower compared with those in the vector control cells (B16/Vec), concomitant with the decreases in both activity and protein level of tyrosinase, a key regulatory enzyme in melanogenesis. Moreover, overexpression of PLD1 resulted in a marked inhibition of melanogenesis induced by alpha-MSH. The inhibition of melanogenesis was well correlated with the decrease in the tyrosinase activity associated with its expression. These results indicated that PLD1 negatively regulated the melanogenic signaling by modulating the expression of tyrosinase in mouse B16 melanoma cells.
Highlights
Phospholipase D (PLD)1 cleaves phosphatidylcholine (PC) in response to a variety of cell stimuli to generate phosphatidic acid (PA) [1,2,3], which acts as a second messenger and can be further converted to other messenger molecules, 1,2-diacylglycerol, and lysoPA [1,2,3]
The inhibition of melanogenesis was well correlated with the decrease in the tyrosinase activity associated with its expression. These results indicated that PLD1 negatively regulated the melanogenic signaling by modulating the expression of tyrosinase in mouse B16 melanoma cells
PLD is considered to be implicated in a variety of cellular responses including cell differentiation [5, 6]
Summary
Materials—L-dopa, [Nle4,D-Phe7]␣-MSH, synthetic melanin, Streptomyces chromofuscus PLD, Clostridium perfringens PLC, the antibody to -actin, and 4-phorbol 12-myristate 13-acetate (PMA) were from Sigma. PLD activity in cell homogenate was measured using the exogenous substrate of phospholipid vesicles. Mixed lipid vesicles (phosphatidylethanolamine/PIP2/egg PC, 10:1.5:1 molar ratio) containing [palmitoyl-3H]dipalmitoylphosphatidylcholine (3 Ci/ml) were added to cell homogenate (50 g protein) in the reaction mixture containing 50 mM HEPES, pH 7.5, 3 mM EGTA, 80 mM KCl, 2.5 mM MgCl2, 2 mM CaCl2, and 1 mM dithiothreitol and were stimulated with 50 M GTP␥S and 100 nM PMA at 37 °C for 1 h in the presence of 0.3% butanol. For the measurement of tyrosinase activity, the cells were washed with ice-cold PBS and lysed by incubating at 4 °C for 30 min in lysis buffer (10 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 10 g/ml leupeptin). The intensity of the bands was quantified by a densitometer
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